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4 N Cocarcinogenic Properties of Nicotine FRED G. BOCK Roswell Park Memorial Institute New York State Department of Health Buffalo, New York 14263 A possible approach toward reduction of smoking-related diseases is the devel- opment of low-tar cigarettes (Gori 1976), a strategy that has been quite successful. Cigarettes delivering less than 15 mg of tar have captured 40% of the market from their high-tar delivery counterparts (Maxwell, this volume). It would appear, however, that many low-tar brands of cigarettes deliver tar with significantly higher nicotine concentrations than do the high-tar brands (Federal Trade Commission 1979). That is, the reduction of nicotine delivery has lagged behind the reduction of' tar. Recent findings that nicotine might contribute to the carcinogenic potency of cigarette smoke may require further evaluation of high- and low-tar cigarettes. There is abundant evidence thai the nicotine in cigarette smoke is a cocarcinogen in concentrations that are found in the condensates we test in the laboratory. One of the chief paradoxes from the Smoking and Health Program has been that, in its first meetings, an advisory group listed all of the compo- nents of cigarette smoke believed to be related to its carcinogenic activity. We hoped to develop a screen to identify high-risk cigarettes on the basis of chemical indicators (Wynder and Hoffmann 1967). In due course various types of experimental cigarettes were tested for long-term mouse skin carcinogenic activity (Gori 1976a, 1976b, 1977; G.B. Gori et al., unpubl. results). In these tests M.R. Guerin assayed each cigarette smoke condensate (CSC) for the suspected indicators. In addition, he also assayed for nicotine because it was expected to indicate the possibility of risks of other diseases. To our surprise the only compound of all of those that M.R. Guerin measured in the cigarettes under bioassay that was consistently related to carcinogenic activity was nicotine, the one compound that no one expected to have a role in the carcinogenic potency of cigarette smoke. TUMOR-PROMOTING ACTIVITY OF TOBACCO EXTRACTS Concurrently with the Smoking and Health Program studies, we examined the 129 4 dI~ r' 3~;K, f r n s 8 .g , t

1301 F.G. Bock tumor-promoting activity of aqueous extracts of unburned tobacco. Female ICR Swiss mice were treated once with a tumor-initiating dose of 125 µg of 7,12-dimethylbenz[a]anthracene (DMBA). They were then treated five times a week with the aqueous extract from 0.5 g of tobacco. One-third of the animals developed tumors within 26 weeks (Bock et al. 1964). We fractionated the crude extract by precipitation with four parts of methanol. Neither the methanol-soluble fraction nor the methanol-insoluble fraction were active (Bock 1968). However, when we recombined the two fractions we recovered the activity at about the level found in the original crude material. Thus, the extract contained at least two materials, both of which were present to provide tumor-promoting activity. We have called these agents copromoters. We subsequently found that the methanol-soluble material was nicotine. The steam distillate of the methanol-soluble fraction contained its copromoting activity, and the copromoting activity of the steam distillate could be duplicated by the nicotine contained in the distillate (Bock and Tso 1976). A paradoxical effect of nicotine is shown by data of the Smoking and Health Program (Gori 1976a; J. Gart, pers. comm.). In that study, each smoke condensate was tested at two dose levels-25 mg and 50 mg per application. An indication of carcinogenic response during the early phases of the experi- ment is the latent period required for 10% of the animals to develop tumors (TQa). The difference in T.0 observed with two dose levels of treatment is therefore a measure of the early dose-response effect of a particular CS C. It can be seen (Fig. 1) that for samples with low concentrations of nicotine, doubling the dose of CSC reduced T,. That is, the latent period was reduced when the dose was doubled. This is the usual dose-response pattern with carcinogenic stimuli. However, for condensates with concentrations of nicotine greater than 5%, an aberrant response was seen. Doubling the dose of CSC and therefore the nicotine, led to a higher T,, i.e., a reduced tumorigenic activity early in the study. CARCINOGENESIS ASSAYS To examine these aspects of the nicotine effect, we conducted a series of cocarcinogenesis assays somewhat similar to those employed by Van Duuren et al. (1976). Solutions of 2 p.g of benzo[a]pyrene (B[a]P) plus 0.12 µg of 12-0-tetradecanoylphorbol-13-acetate (TPA) in 0.2 ml of acetone behaved about the same as 25-50% CSC in mouse skin carcinogenesis assays (Bock et al. 1974). Four test solutions containing this mixture plus zero to 1.2 mg of nicotine were then tested in a conventional mouse skin carcinogenesis bioassay. The biological effectiveness of the high levels of nicotine was about the same as a 50% solution of CSC from a high-nicotine cigarette. This was indicated by ~ the fact that about 15% of the mice died within 10 weeks due to nicotine toxicity. With only slightly more nicotine, e.g., 1.3 mg per dose, 35% of the !~ . 80 40 ~ F-~ II' a 0 -40 -80 I 2 , 3 Cocarcinogenic Properties of Nicotine /131 I , I. I 4 5 Conc. Nicotine d % ) 0 6 0 7 Figure 1 Dependence of dose-response effects on nicotine concentration. The data of Gori et al. (1976a) has been used to determine the increase in time required for 10% of the animals to develop tumors (ATy,o, in days) when the dose of cigarette smoke condensate was reduced by 50%. ( ) Regression line for all of the points; (-) points omitting the four extreme nicotine concentrations, below 3% and above 7%; r, respective linear correlation coefficients_ mice died within 10 weeks. As with smoke condensates, toxicity of the test solutions was observed primarily in the first few weeks of study. The results of the assays (Fig. 2) showed two effects of nicotine: en- hancement and early inhibition of carcinogenic activity. Moderate levels of nicotine (0.3-0.6 mg per dose) increased the activity of the B[a]P-TPA mixture. Similar results have been obtained in two subsequent experiments. Increasing the dose to 1.2 mg per application led to a marked delay in tumor appearance. This reduction remained after correction for nicotine toxicity. We must now determine whether these observations have meaning for the smoker. Experiments reported many years ago by Boutwell (1964) require us to be very cautious when extrapolating from mouse to man. Boutwell applied a 1.5-mg dose of croton oil to three groups of mice, but used a different dosage regimen for each group. To one group, he applied the material at the rate of 125 f.tg once a week; the second group was given 31 fr.g twice a week until a total dose of 1.5 mg was applied; the third group was given 6.25 µg ten times a week. The first two groups developed abundant numbers of tumors. But the latter group receiving the same total dose of croton oil developed very few COos:~zs%oz

132 / F.G. Bock Figure 2 Effect of nicotine on the carcinogenic activity of a mixture of Bla ] P plus TPA. Mice were treated with 10 jig B[a]P plus 0.6 µg TPA in acetone solutions containing the nicotine levels indicated on the abscissa. The probabilities of tumor appearance (1-P) at three staQes of the experiment are indicated by the respective curves. tumors. It appeared that there was some minimum individual dose required for expression of tumor-promoting activity. In the long-term mouse skin assays of CSC, we apply nearly lethal doses of nicotine once or twice a day. This is quite different from applying 200-300 times daily a dose yielding only minimally detectable physiological effects. The latter regimen, which may resemble the low-dose, high-frequency exposure of Boutwell's third group is typical of the usual human experience. We can see that, if nicotine has the same cocarcinogenesis mechanism as croton oil, the experiments we conduct in the laboratory may overestimate the possible hazard of nicotine to the smoker. Until we understand the mechanism by which nicotine acts as a cocarcinogen, we cannot determine whether our observations can be extrapolated to the human situation and whether, indeed, cigarettes with relatively high nicotine levels would have enhanced carcinogenic activity for man. If our data do not relate to man, a relatively high-nicotine, low-tar cigarette would permit the strategy of getting rid of most of the unknown cocarcinogens in smoke while retaining sufficient smoke acceptability so that more hazardous products can be supplanted. THE MECHANISM OF NICOTINE'S ACTIVITY Knowing that nicotine acts to enhance the carcinogenic activity of B[a]P-TPA mixtures, we may ask if nicotine acts in concert with TPA alone, with B[a]P alone, or with both together. In other words, does nicotine enhance tumor x promotion, does it enhance initiation, or does it act through some other mechanism? Cocarcinogenic Properties of Nicotine / 133 To answer this question, we painted 90 female ICR Swiss mice once with 150 µg of B[a]P in 0.2 ml acetone at 63-69 days of age. After 3 weeks, they were treated five times a week for 26 weeks with 0.2 ml of acetone containing 0.6 F,tg of TPA. These mice served as controls. A second group of mice was treated with the B[cr]P solution containing 3 mg of nicotine/ml. These animals were then treated with TPA just as the controls. The third group of mice was treated with B[a]P as were the controls, but were subsequently painted with the TPA solution containing 3 mg of nicotine/ml. Negative control groups of 50 mice were treated with either B[a]P or B[a]P plus nicotine followed by acetone, or with acetone followed by either TPA or TPA plus nicotine. The results (Fig. 3) showed no effect of nicotine on either initiation or promotion. One tumor was observed in the negative control group treated with B[a]P followed by acetone. All of the tumors were benign. Although the nicotine in this experiment (3 mg/m]) yielded an optimal response in our earlier cocarcinogenesis experiments, it did not add to the activity of either B[a]P or TPA when added to either of these agents sepa- rately. In the cocarcinogenesis experiment, however, the level of TPA was low, 0.6 µg/ml in contrast to 3 µg/ml in the present initiation-promotion study. Could nicotine be effective only with low doses of TPA? We conducted a second experiment in which mice were initiated by a single application of 125 p.g of DMBA in 0.25 ml of acetone and then painted five times a week for 34 weeks with 0.2 ml of an acetone solution of TPA or TPA plus nicotine. The first group of 24 mice was treated with 2 µg/ml TPA solution as positive 60 240 ~ 3 0 L-Y Figure 3 Effect of nicotine on initiation and promotio, (.) 150 jig B[a IP once followed live times a week with 0.6 jig TPA; (o) 150 µg Bla ] P plus 0.6 mg nicotine once followed five times a week with 0.6 pg TPA: (-) 150 µg B[a[P once followed five times a week with 0.6 µg TPA plus 0.6 mg nicotine. ~ola~s~z~zaz

134 / F.G. Bock Table 1 Effect of Nicotine on Tumor Prom otion Tumor incidence" Promoting stimulus' Number of mice number Percent 2 µg/ml TPA 24 14 58 0.5 µrJml TPA 96 5 5 0.5 µg/ml TPA plus 3 mg/mi nicotine 96 8 8 'Mice were treated once with 125 I.cg DMBA in 0.25 ml of acetone followed, after 3 wect;s,l by 5ve applications weekly of 0.2 ml of the respective materials in acetone. bAt 34 weeks, all of the tumors were benign. controls. The second group of 96 mice was treated with 0.5 ftg of TPA/ml acetone, a dose earlier found to have minimal promoting activity. The third group of 96 mice was treated with 0.5 f.t.g/ml of TPA plus 3 mg nicotine/ml acetone. The results (Table 1) showed that nicotine does not enhance the activity of low levels of TPA when this agent is applied in an initiation-promotion sequence. The answer to our first question, therefore, is that nicotine acts throuQh some mechanism other than the classical initiation-promotion se- quence. Is the nicotine effect due to the alkaloid itself or to one of its identified metabolites? Nicotine is metabolized into a number of products, among which ~ both cotinine and nicotine 1'-N oxide (NNO) have been suspected to be - carcinogenic (Boyland 1968; Gorrod and Jenner 1975). One or both of these metabolites might serve as a cocarcinogen with the B[a]P-TPA mixture. To evaluate this possibility, we prepared a solution containing 10 f.tg of B[a]P and 0.6 µg of TPA per ml of acetone. This mixture is a moderate carcinogenic stimulus in our studies and its activity was known to be enhanced by nicotine. Either nicotine (2.5 or 5 mg/ml), cotinine (2.5 or 10 mg/ml), or NNO (2.5 or 10 mg/ml) was added to provide six experimental test solutions. Groups of 45-75 ICR Swiss female mice (Table 2) were painted ten times a week for 39 weeks with 0.2 ml of the respective test solutions or with the control solution containing B[a]P and TPA but no additive. The tumor incidence was recorded weekly and the statistical significance of differences in incidence was deter- mined using a chi-square method (Peto 1974; Gart 1975). The results showed that both 2.5 and 5 mg of nicotine/ml B[a]P-TPA solution caused a substantial and significant enhancement of carcinogenic ac- tivity (Fig. 4). There was no difference in effect between the two nicotine doses. Because we observed earlier that a 6 mg/mI dose of nicotine caused early inhibition of carcinogenesis, we conclude that the optimal level of 'nicotine for carcinogenesis enhancement lies between 2.5 and 5 mg/ml. E E ~ o z U t0 U C Q. I g° _-;080 b V e: V V oo 10 oo N ~ I N OG .~ ct M O v ~ - - ri rn !n o0 00 00 0 N - - .-. .~ G U v DO t- Cl oo t x" .O (V I- N fV ~ .o E r ~ - ~O U O N an M C^ N N^ .C] 7 c y 1 O~~ h V"1 h Y r"nr:vvvv 8 Z ° 3 ~ v) o CD wl !n ~n ~n rv-) v% vvvv 0 ~ aw o ~ o ~ 5 = O ~a 3 0 ~ . u c Z 0 •_ ° ~ zz F o '= z u 1 ~ E E 3 ~o ~ ~ E _~b E Eb E 00 E-u) E bn E en C 0 E~n E~n E ~ N v1 N~ N h U O ~,++++-r+ N pl~_ 6l ~ Z N 0 0 ~ Q~ Q Q Q Q [-h FF^HhE- .g m E 7~ _d U ~ - 4 L G. G. G G z_z ~ W F- ~ 21 ~ q CO q q ~~%~~%oz

1361 F.G. Bock . 100 Bo N 60 C F 3 40 ~ ~ 20 0~1 O O 0 0 0 0 ~ O 0 o---o'~ Figure 4 Effect of nicotine and NNO on the carcinogenic activity of a mixture of B[a]P plus TPA. Mice were treated ten times a week with 0.2 ml of acetone containing 10 µg B[a]P plus 0.6 µg TPA/mI plus. (.) B[a]P plus TPA alone; (o) 2.5 mg nicotine/ml; (o) 5 mg nieotine/ml; (o) 2.5 mg NNOIml; (x) 5 mg NNOlmI. There were more tumors in the mice painted with cotinine than in the controls (Table 2). The differences, however, were not sufficiently great to suggest that metabolic conversion of nicotine to cotinine could account for the cocarcinogenic activity of nicotine. Mice treated with solutions containing NNO developed about half as many tumors as did the controls. The effect was statistically significant and appeared to be more striking during the first 30 weeks of study (Fig. 4). Metabolism of nicotine to NNO might well account for the early inhibition of B[a]P-TPA carcinogenesis by high doses of nicotine. We plan to explore this effect further by testing lower levels of NNO in the B[a]P-TPA mixture. CONCLUSIONS In summary, the results of these experiments show that the enhancement of B[a]P-TPA carcinogenesis by nicotine is not due to a specific effect of the alkaloid on either initiation or promotion. Furthermore the enhancement of carcinogenesis is not a consequence of the metabolic conversion of nicotine to either cotinine or NNO. If metabolism is critical for nicotine activity, some other metabolite, not yet suspected to be carcinogenic, must be involved. The inhibitory effect of high doses of nicotine on early stages of B[cr]P- TPA carcinogenesis may be due to metabolic production of NNO. In this respect, it is of interest that NNO reduces the toxicity of nicotine in mice (Barrass et al. 1969). High levels of NNO have only a moderate inhibitory effect on tumorigenesis. This observation is parallel to our experience with Cocarcinogenic Properties of Nicotine 1137 nicotine (Fig. 2); the inhibitory effect was moderate and was less apparent late in the experiments. We are left with our original question of whether nicotine could be a cocarcinogen for human cigarette smokers. Boutwel]'s data suggest a threshold dose for tumor promotion because repeated doses of a subthreshold level of promoter are ineffective. If the action of nicotine on tumorigenesis were to enhance the promoting effect of TPA, we might question its importance as a cocarcinogenic stimulus for smokers. This is not the case, however. There is, as yet, no laboratory evidence of a threshold for cocarcinogenesis by nicotine. Until we understand the mode of action of this alkaloid in mouse skin bioas- says, we must consider nicotine a candidate of human carcinogenic hazard. ACKNOWLEDGMENTS I am grateful to Helen W. Fox and Huston K. Myers who provided technical assistance for the studies reported here. REFERENCES Barrass, B.C., J. W. Blackburn, R.W. Brimblecombe, and P. Rich. 1969. Modification of nicotine toxicity by pretreatment with different drugs. Biochem. Phannacol. 18:2145. Bock, F. G. 1968. The nature of tumor-promoting agents in tobacco products. Cancer Res. 28:2363. Bock, F.G. and T.C. Tso. 1976. Chemical and biological identification of tumorigenic components of tobacco. In Proceedings of the Third World Conference on Smoking and Health (ed. E.L. Wynder, D. Hoffmann, and G.B. Gori), vol. 1, p. 161. DHEW publication number(NIH) 76-1221. Government Printing Office, Washing- ton, D.C. Bock, F.G., G.E. Moore, and S.K. Crouch. 1964. Tumor-promoting activity of extracts of unburned tobacco. Science 145:831. Bock, F.G., I.D.J. Gross, and R.L. Priore. 1974. Synergistic action ofbenzo[a]pyrene, tetradecanoyl phorbol acetate when applied concurrently. Abstr. Ilth Inr. Cancer Conf. 2:43. Boutwell, R.K. 1964. Some biological aspects of skin carcinogenesis. Progr. Exp. Tianor Res. 4:207. Boyland, E. 1968. The possible carcinogenic action of alkaloids of tobacco and betel nut. Planta Medica Suppl. 11: 13. Federal Trade Commission. 1979. Report of "tar" and nicmine contentof the smoke of 176 rarieties of cigarettes. Government Printing Office, Washington, D. C. Gart, J.J. 1975. Letter to the editor. Brit. J. Cancer 31:696. Gori, G.B. 1976. Low-risk cigarettes: A prescription. Science 194:1243. Gori, G.B. (ed.). 1976a. Report No. 1. Totvard a less hazardous cigarette. Thefirst set of e_rperimental cigarettes. DHEW publication number (NIH) 76-905. Government Printing Office, Washington, D.C. 1976b. Report No. ?. Totrard a less hazardous cigarette. The second set of

1381 F.G. Bock experimental cigarettes. DHEW publication number (NIH) 76-1111. Government Printing Office, Washington, D.C. . 1977. Report No. 3. Toward a less hazardous cigarette. The third set of experimental cigarettes. DHEW publication number (NIH) 77-1280. Government Printing Office; Washington, D.C. Gorrod, J.W. and P. Jenner. 1975. The metabolism of tobacco alkaloids. Essa_rs in Toxicol. 6:35. Peto, R. 1974. Guidelines on the analysis of tumor rates and death rates in experimental animals. Brit. J. Cancer 29:101. Van Duuren, B.L. and B.M. Goldschmidt. 1976. Cocarcinogenic and tumor-promoting agents in tobacco carcinogenesis. J. Nail. Cancer Inst. 56:1237. Wynder, E.L. and D. Hoffmann. 1967. Tobacco and tobacco smoke. Studies in experimental carcinogenesis. Academic Press, New York. 11 COMMENTS HOFFMANN: Do you have any evidence as to the mechanism underlying your hypotheses, that a cocarcinogenic effect or inhibitory effect may be due to metabolites of nicotine formed upon its application to mouse skin? Do you have any evidence that topical or subcutaneous administration of nicotine in mouse skin leads directly to NNO respectively to cotinine? Isn't this what happens in the liver? BOCK: One can say that the major metabolism occurs in the liver, but that does not necessarily preclude the metabolism of small amounts in the skin. It wasn't that we felt nicotine metabolism was the mechanism for its action, but that we wanted to determine whether we could rule it out. We can certainly rule out cotinine as an intermediate for the nicotine effects. If all the nicotine that passed through the skin were converted to cotinine, we would still have tested four times that level. Cotinine is clearly not involved. I seriously doubt whether NNO is involved, but it could be involved as a tumor inhibitor. It certainly is not responsible for the tumor-enhancing properties. WYNDER: It is also becoming evident from pathological data that smokers of low-tar, low-nicotine products have fewer bronchial lesions. Therefore, the human data would suggest that low doses of nicotine do not act to enhance tumorigenic activity, at least in the lung. I wonder how you think this falls in line with the data you presented? BOCK: I believe that high nicotine would be responsible for some of the human tumors we've seen in the past. The tumor inhibition we see is a consequence of extremely high levels that wouldn't be observed in human smoking. GORI: What he says is not incompatible with what we're seeing. WYNDER: With the higher dose, of course, you increase the toxicity, and in fact, we did an experiment like yours years ago with high-nicotine tars. You get so Cocarcinogenic Properties of Nicotine 1139 much toxicity that it is difficult to evaluate the weight curves. I guess you studied the weight of these animals BOCK: The animals with high nicotine dosage have less weight than the controls or those with lower nicotine exposures. In terms of survival, with the highest levels of nicotine, we usually start with perhaps 150 animals per group, and 70 or so survive until they develop a tumor oruntil the end of the experiment. GORI: Part of the question Dietrich [Hoffmann] raised is the idea of having low-tar and high-nicotine cigarettes. Part of the problem disappears when we stop to realize that whenever we advocate such change in ratios we are still speaking of a much lower absoltite level of nicotine than what we have in cigarette smoke today. We are speaking of cigarettes on the order of 5 mg of tar, or less, and on the order of 0.6-0.7 mg nicotine. In my opinion the differences that we are talking about are not so relevant, because delivery of low nicotine from low-tar cigarettes is still much lower that what the average intake of nicotine is today. BOCK: I wish I could agree with you without reservation, Gio [Gori], but the problem when you're dealing with nicotine is that it's not the amount of nicotine that's in the condensate, it's the availability of the nicotine to the tissues of the smoker. If we measure availability of nicotine to mouse skin, by mouse mortality, we find that about 20 mg nicotine/ml aqueous cigarette extract gives you a 50% mortality. The same effect is produced by an acetone solution of cigarette smoke condensate containing 12- 14 mg nicotine/m1, and by a pure solution of about 6 mg nicotine/ml. So you have a threefold range of nicotine toxicity, depending on the vehicle on which it is contained. If we get rid of half of the tar, we might be doubling the relative availability of nicotine that's contained in that tar. I just don't know. WYNDER: This is a very important point, in relation to cardiovascular disease problems. The tar-to-nicotine ratio has changed dramatically during the last 2 years-from 20:1, to 10:1, to 8:1-and I think this concept is very important in terms of cardiovascular disease development. GORt: I want to correct the impression that the ratio has not changed in the last few years. HOFFMANN: The ratio has increased in favor of nicotine. There is no question about it. Nicotine concentration in "tars" used to be 6%. In the low-tar cigarette smoke condensate it is now 8-10%.